Microcore samples, small wood pieces taken out from trees are a commonly used tool for studying wood anatomy.
One approach to obtain microcores is the Trephor tool, as an effective way to take out microcores with 2 mm diameter . Once the microcores samples are taken out, they must be conserved in formaldehyde alcohol acetic acid solution (FAA), usually in Eppendorf, for a maximum of two weeks. Afterward, the FAA needs to be replaced with 70% ethanol, and the samples need to be stored at 4°C, and processed as soon as possible. The processing includes embedding the microcore samples in paraffin. The microcores must not dry when we work with them, so continuous dipping in ethanol at this stage is recommended. Looking at the woody part of the microcores under a light, we mark the transverse orientation on each microcore before putting them in marked bio-cassettes. Once in the cassette, the microcores are immediately put in ethanol. From there, by using the machine, the microcores undergo a 12-step process of dehydration with ethanol and D-limonene, before they are finally immersed in liquid paraffin at 65°C. One at a time, the metal plates are used to embed the microcores to the back of the cassettes with liquid paraffin. Afterward, they are placed in the freezer to harden for a minimum of 20 minutes, until the paraffin has completely solidified and the cassette with the attached sample can be taken out of the metal tray.
Once hardened, the paraffin blocks were trimmed to expose the wood and immersed in tap water immersion (at room temperature for 16-48 hours) for the wood to soften. Microcore sections are commonly cut by rotary microtomes (e.g. Leica Microsystems). A stripe of microcores sections are put in room temperature water, and then in a water bath from where they are 'fished out' with microscope slides that were previously treated with albumin, allowing for better adhesion. The slides are then dried at 75°C for 20 minutes and the residual paraffin is cleaned by gradual immersion in D-limonene and ethanol. Both safranin (Merck, Darmstadt, Germany) (0.04%) and astra blue (Sigma-Aldrich, Steinheim, Germany) (0.15%) water mixture can be used for coloring the microcore sections, but this depends on the goals and what aims to be observed. By using suitable glue, such as Euparal, and cover glass, permanent microcore microscope slides are obtained. Once the glue dries, they can be observed under a microscope and if needed, images can be taken and further analyzed.
A visual presentation of the process of the whole process is presented in this short video.
- Rossi, S., Anfodillo, T., & Menardi, R. (2006). Trephor: a new tool for sampling microcores from tree stems. Iawa Journal, 27(1), 89-97.